The primary goal of the proposed investigation is a detailed analysis of the cellular and molecular aspects of localization of two pseudomonas aeruginosa virulence factors: exotoxin A and the pilus adhesin. Biochemical and genetic tools will be used to identify the pathway for the export of these proteins. The export pathway will be studied by examining the various steps of cytoplasmic membrane traversal and translocation across the outer membrane. Techniques of protein fusion and site directed mutagenesis will be used to identify domains in exotoxin A and pilin, that play a role in various steps of membrane translocation. Mutants of P. aeruginosa defective in export of exotoxin A and pilin will be identified and characterized. Defective genes in such mutants will be complemented by recombinant plasmids, and coding sequences for the export genes will be isolated from such plasmids. These studies should not only help in a better understanding of regulation of biosynthesis of virulence factors by P. aeruginosa, but also open new lines of investigation into the mechanism of extracellular protein localization by Gram-negative bacteria.